Functional characterization of spectrin-actin-binding domains in 4.1 family of proteins
Functional characterization of spectrin-actin-binding domains in 4.1 family of proteins
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DOI：
10.1021/bi0256330
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发表时间：
2002-06-11
期刊：
影响因子：
2.9
通讯作者：
Mohandas, N
中科院分区：
文献类型：
--
作者：
Gimm, JA;An, XL;Mohandas, N
蛋白质 4.1R 是蛋白质家族的典型成员，该家族包括 4.1G、4.1B 和 4.1N。 4.1R 通过其血影蛋白-肌动蛋白结合 (SAB) 结构域与血影蛋白和肌动蛋白协同结合，在维持膜机械完整性方面发挥着至关重要的作用。虽然 4.1R 和血影蛋白之间的二元相互作用已得到很好的表征，但 4.1R 中的肌动蛋白结合位点仍未确定。此外，对于 4.1R 同源物与血影蛋白和肌动蛋白的相互作用知之甚少。在本研究中，我们表明 4.1R 的 10 kDa 血影蛋白-肌动蛋白结合域内的 8 个氨基酸基序 (LKKNFMES) 在 4.1R 与肌动蛋白的结合中发挥着关键作用。该基序发生突变的重组 4.1R SAB 结构域肽与血影蛋白和肌动蛋白形成三元复合物的能力显着降低。二元蛋白质-蛋白质相互作用研究表明，这种减少是由于突变型 SAB 肽无法与肌动蛋白丝结合，而对血影蛋白的亲和力却没有改变。我们还证明，外显子 16 编码的 21 个氨基酸盒的 14 个 C 端残基与外显子 17 编码的残基 27-43 一起构成了功能齐全的最小血影蛋白结合基序。最后，我们发现4.1N SAB结构域无法与血影蛋白和肌动蛋白形成三元复合物，而4.1G和4.1B SAB结构域能够形成这样的复合物，但效率低于4.1R SAB。这是由于 4.1G 和 4.1B SAB 结构域与肌动蛋白相互作用的能力下降，但与血影蛋白不相互作用。这些数据使我们能够提出 4.1R-血影蛋白-肌动蛋白三元复合物的模型，该模型可以作为调节各种细胞类型中基于血影蛋白的细胞骨架相互作用的通用范例。
Protein 4.1R is the prototypical member of a protein family that includes 4.1G, 4.1B, and 4.1N. 4.1R plays a crucial role in maintaining membrane mechanical integrity by binding cooperatively to spectrin and actin through its spectrin-actin-binding (SAB) domain. While the binary interaction between 4.1R and spectrin has been well characterized, the actin binding site in 4.1R remains unidentified. Moreover, little is known about the interaction of 4.1R homologues with spectrin and actin. In the present study, we showed that the 8 aa motif (LKKNFMES) within the 10 kDa spectrin-actin-binding domain of 4.1R plays a critical role in binding of 4.1R to actin. Recombinant 4.1R SAB domain peptides with mutations in this motif showed a marked decrease in their ability to form ternary complexes with spectrin and actin. Binary protein-protein interaction studies revealed that this decrease resulted from the inability of mutant SAB peptides to bind to actin filaments while affinity for spectrin was unchanged. We also documented that the 14 C-terminal residues of the 21 amino acid cassette encoded by exon 16 in conjunction with residues 27-43 encoded by exon 17 constituted a fully functional minimal spectrin-binding motif. Finally, we showed that 4.1N SAB domain was unable to form a ternary complex with spectrin and actin, while 4.1G and 4.1B SAB domains were able to form such a complex but less efficiently than 4.1R SAB. This was due to a decrease in the ability of 4.1G and 4.1B SAB domain to interact with actin but not with spectrin. These data enabled us to propose a model for the 4.1R-spectrin-actin ternary complex which may serve as a general paradigm for regulation of spectrin-based cytoskeleton interaction in various cell types.